Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography

In the last few years, SAXS of biological materials has been rapidly evolving and promises to move structural analysis to a new level. Recent innovations in SAXS data analysis allow ab initio shape predictions of proteins in solution. Furthermore, experimental scattering data can be compared to calculated scattering curves from the growing data base of solved structures and also identify aggregation and unfolded proteins. Combining SAXS results with atomic resolution structures enables detailed characterizations in solution of mass, radius, conformations, assembly, and shape changes associated with protein folding and functions. SAXS can efficiently reveal the spatial organization of protein domains, including domains missing from or disordered in known crystal structures, and establish cofactor or substrate-induced conformational changes. For flexible domains or unstructured regions that are not amenable for study by many other structural techniques, SAXS provides a unique technology. Here, we present SAXS shape predictions for PCNA that accurately predict a trimeric ring assembly and for a full-length DNA repair glycosylase with a large unstructured region. These new results in combination with illustrative published data show how SAXS combined with high resolution crystal structures efficiently establishes architectures, assemblies, conformations, and unstructured regions for proteins and protein complexes in solution.     

Spinal loading during manual materials handling in a kneeling posture.

Stooped, restricted, kneeling, and other awkward postures adopted during manual materials handling have frequently been associated with LBP onset. However, lift assessment tools have focused on materials handling performed in an upright, or nearly upright standing posture. Unfortunately, many of the tools designed to analyze standing postures are not easily adapted to jobs requiring restricted postures. Therefore, the objective of this study was to evaluate spinal loading during manual materials handing in kneeling postures and determine if those loads can be predicted using simple regression. An EMG-driven biomechanical model, previously validated for upright lifting, was adapted for use in kneeling tasks. Subjects knelt under a 1.07m ceiling and lifted luggage of six weights (6.8, 10.9, 15.0, 19.1, 23.1 and, 27.2kgf) to one of four destination heights (0, 25.4, 53.3, 78.7cm). Spine loading was significantly affected by both destination height and load weight. Destination height increased compression, AP shear and lateral shear by an average of 14.5, 3.7 and 6.6N respectively per cm height increase. Load weight increased compression, AP shear and lateral shear by an average of 83.8, 27.0 and 13.1N respectively per kgf lifted. Regression equations were developed to predict peak spine loading using subject height, load weight and destination height with R(2) values of 0.62, 0.51 and 0.57 for compression, AP and lateral shear respectively.     

Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors

Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of mu-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut-similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treatment of abdominal pain and irritable bowel syndrome.     

The Amazonas‐trap: a new method for sampling plant‐inhabiting arthropod communities in tropical forest understory

Methods to quantify plant‐insect interactions in tropical forests may miss many important arthropods and can be time consuming and uneven in capture efficiency. We describe the Amazonas‐trap, a new method that rapidly envelops the target plant for sampling arthropods. We evaluated the efficiency of the Amazonas‐trap by comparing it with two commonly used sampling methods to collect arthropods from plants: the beating tray and manual collection. Samples were collected in 10 permanent plots, in the Ducke forest reserve, Manaus (Amazonas, Brazil). In each plot we sampled 18 plant individuals of Protium sp. (Burseraceae): six by a beating tray, six by manual collection, and six using the Amazonas‐trap. All insects were identified to the family level and those belonging to the order Hymenoptera were identified to the species and morphospecies level. The new method sampled more insect families and more Hymenoptera species than tree beating and manual collection. Of the 75 total families collected, 20 were sampled exclusively by the Amazonas‐trap, seven were only collected with a beating tray, and seven were sampled exclusively with manual collecting. A similar pattern was found for abundance: Amazonas‐trap sampled more individuals, followed by the beating tray and manual collection. Small and winged arthropods were more abundant in Amazonas‐trap, explaining the highest richness of Hymenoptera and insect families sampled with this method. The new method sampled more spiders, wood‐fungi feeders, sap suckers, omnivorous, parasitoids, and insect predators than the other methods, but was equally effective in sampling leaf‐feeders and ants. Amazonas‐trap was more time consuming in the field, but for all diversity parameters evaluated, the new method showed better performance for collecting invertebrates on plants.     

The Antarctic psychrophiles Chlamydomonas spp. UWO241 and ICE-MDV exhibit differential restructuring of photosystem I in response to iron

Chlamydomonas sp. UWO241 is a psychrophilic alga isolated from the deep photic zone of a perennially ice-covered Antarctic lake (east lobe Lake Bonney, ELB). Past studies have shown that C. sp. UWO241 exhibits constitutive downregulation of photosystem I (PSI) and high rates of PSI-associated cyclic electron flow (CEF). Iron levels in ELB are in the nanomolar range leading us to hypothesize that the unusual PSI phenotype of C. sp. UWO241 could be a response to chronic Fe-deficiency. We studied the impact of Fe availability in C. sp. UWO241, a mesophile, C. reinhardtii SAG11-32c, as well as a psychrophile isolated from the shallow photic zone of ELB, Chlamydomonas sp. ICE-MDV. Under Fe-deficiency, PsaA abundance and levels of photooxidizable P700 (ΔA₈₂₀/A₈₂₀) were reduced in both psychrophiles relative to the mesophile. Upon increasing Fe, C. sp. ICE-MDV and C. reinhardtii exhibited restoration of PSI function, while C. sp. UWO241 exhibited only moderate changes in PSI activity and lacked almost all LHCI proteins. Relative to Fe-excess conditions (200 µM Fe²⁺), C. sp. UWO241 grown in 18 µM Fe²⁺ exhibited downregulation of light harvesting and photosystem core proteins, as well as upregulation of a bestrophin-like anion channel protein and two CEF-associated proteins (NdsS, PGL1). Key enzymes of starch synthesis and shikimate biosynthesis were also upregulated. We conclude that in response to variable Fe availability, the psychrophile C. sp. UWO241 exhibits physiological plasticity which includes restructuring of the photochemical apparatus, increased PSI-associated CEF, and shifts in downstream carbon metabolism toward storage carbon and secondary stress metabolites.

     

Within-Farm Changes in Dairy Farm-Associated Salmonella Subtypes and Comparison to Human Clinical Isolates in Michigan, 2000-2001 and 2009

Temporal changes in the distribution of Salmonella subtypes in livestock populations may have important impacts on human health. The first objective of this research was to determine the within-farm changes in the population of subtypes of Salmonella on Michigan dairy farms that were sampled longitudinally in 2000-2001 and again in 2009. The second objective was to determine the yearly frequency (2001 through 2012) of reported human illnesses in Michigan associated with the same subtypes. Comparable sampling techniques were used to collect fecal and environmental samples from the same 18 Michigan dairy farms in 2000-2001 and 2009. Serotypes, multilocus sequence types (STs), and pulsed-field gel electrophoresis (PFGE) banding patterns were identified for isolates from 6 farms where >1 Salmonella isolate was recovered in both 2000-2001 and 2009. The distribution of STs was significantly different between time frames (P < 0.05); only two of 31 PFGE patterns were identified in both time frames, and each was recovered from the same farm in each time frame. Previously reported within-farm decreases in the frequency of multidrug-resistant (MDR) Salmonella were due to recovery of MDR subtypes of S. enterica serotypes Senftenberg and Typhimurium in 2000-2001 and genetically distinct, pansusceptible subtypes of the same serotypes in 2009. The annual frequency of human illnesses between 2001 and 2012 with a PFGE pattern matching a bovine strain decreased for patterns recovered from dairy farms in 2000-2001 and increased for patterns recovered in 2009. These data suggest important changes in the population of Salmonella on dairy farms and in the frequency of human illnesses associated with cattle-derived subtypes.